Kit Components

Item	Quantity	Storage
Pre-coat 96 well microplate	12 x 8 well strips	+4°C
Lyopholized standard	2 vials	+4°C
Sample dilution buffer	20 ml	+4°C
Biotinylated detection antibody	120 µl	+4°C
Antibody dilution buffer	10 ml	+4°C
HRP-streptavidin conjugate	120 µl	+4°C
SABC dilution buffer	10 ml	+4°C
TMB substrate	10 ml	+4°C
Stop solution	10 ml	+4°C
Wash buffer (25X)	30 ml	+4°C
Plate sealers	5 adhesive strips	+4°C

Required materials

The following materials are not included in the kit, but are required to run this assay:

• Microplate reader capable of measuring absorbance at 450nm ± 10nm.
• Squirt bottle, multichannel pipette reservoir, or automated microplate washer.
• Graph paper or computer software capable of generating logarithmic functions.
• Test tubes and Eppendorf tubes to prepare standards and samples.
• Multi- and single-channel pipettes and sterile pipette tips.
• Deionized or distilled water.
• Absorbent paper towels.
• 37°C incubator.

Important notes

• Sample concentrations should be estimated before being used in the assay and a proper dilution factor should be selected to make the diluted target protein concentration fall in the optimal detection range of this kit.
• Sample matrix components will interfere with the test results. All samples must be diluted at least 1:2 with Sample Dilution Buffer.
• Hemolysis can greatly impact the validity of test results. Take care to minimize hemolysis.

Bring all reagents and samples to room temperature before use.

We recommend that all standards and samples be assayed in duplicate.

• Step 1

  Prepare all reagents, samples, and working standards as directed in the previous sections.

• Step 2

  Determine the number of microplate strips required for the assay and insert them into the plate frame. Unused strips should be stored in the foil pouch at +4°C.

• Step 3

  Add 100μl of standard solutions to the standard wells. Note: A plate layout is provided to record standards and samples assayed.

• Step 4

  Add 100μl of samples to the sample wells. Note: All samples must be properly diluted in order to produce sample values within the dynamic range of the assay. All samples must be diluted at least 1:2 with Sample Dilution Buffer.

• Step 5

  Cover with an adhesive plate sealer provided and incubate at 37°C for 90 minutes.

• Step 6

  Remove the plate sealer, aspirate the liquid from the plate, and wash the plate two times with Wash Buffer. Soak wells with 350μl of Wash Buffer for 1-2 minutes each time. Note: Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Blot the plate onto paper towels or other absorbent material. Do not let the wells dry completely at any time!

• Step 7

  Add 100μl of Biotinylated Detection Antibody working solution to each well.

• Step 8

  Cover with a new adhesive plate sealer and incubate at 37°C for 60 minutes.

• Step 9

  Remove the plate sealer, aspirate the liquid from the plate, and wash the plate three times with Wash Buffer. Soak wells with 350μl of Wash Buffer for 1-2 minutes each time.

• Step 10

  Add 100μl of HRP-Streptavidin Conjugate (SABC) working solution to each well.

• Step 11

  Cover with a new adhesive plate sealer and incubate at 37°C for 60 minutes.

• Step 12

  Remove the plate sealer, aspirate the liquid from the plate, and wash the plate five times with Wash Buffer. Soak wells with 350μl of Wash Buffer for 1-2 minutes each time.

• Step 13

  Add 90μl of TMB Substrate to each well. Cover with a new adhesive plate sealer and incubate at 37°C in the dark for 10-20 minutes. Note: This incubation time is for reference only, the optimal reaction time should be determined by the end-user and can be shortened or extended according to the actual color change. The reaction may be terminated when a gradient is apparent in the standard wells. Do not exceed 30 minutes!

• Step 14

  Add 50μl of Stop Solution to each well. The color in the wells should change from blue to yellow immediately. Note: If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.

• Step 15

  Determine the optical density (O.D. value) of each well immediately using a microplate reader set to 450 nm.